The long term objective of the research proposed is the explication of the distribution, dynamics and function of cholesterol in mammalian cell membranes. Although cholesterol is a major constituent of plasma membrances, its precise disposition and function are to a large degree unknown. The mechanisms of cholesterol transfer between tissues and plasma and between biological membrances ae unknown. The literature contains proposals for mechanisms of unmediated transfer which fall into two categories: diffusion through the aqueous phase and transfer during the formation of a collision complex. These mechanisms can be distinguished from the dependence of transfer rate on concentration of donor and acceptor. We shall examine the exchange of [3H]-cholesterol between red cells themselves and between red cells, sonicated liposomes and plasma lipoproteins to determine the mechanism of cholesterol transfer. We shall use cholesterol oxidase, an enzyme which catalyzes cholesterol oxidation, as a probe to study cholesterol distribution and transfer between the plasma membrane and internal organelles of cultured cells. Our previous studies showed that cholesterol enrichment of red cells to just above the physiological levels leads to membrane reorganization. A study of the molecular basis of this change is proposed. We showed previously that cholesterol transmembrane movement in red cells is extremely rapid. We now propose studies at subzero temperature to examine how rapid this motion is and to determine the transbilayer distribution of cholesterol in the red cell membrane. The rapid transmembrane movement of cholesterol may lie at the heart of its function. We shall examine the hypothesis that redistribution of cholesterol across the membrane occurs in response to stress such as that imposed by shape deformation or the incorporation at the outer membrane leaflet of an exogenous molecule such as lysolecithin. In these studies the antibiotic filipin, which forms complexes with sterols, will be used as a probe for cholesterol distribution.